We are currently characterizing the abnormalities for regulation of human muscle glycogen synthesis in insulin-resistant subjects. In insulin- resistant subjects, fasting glycogen synthase phosphatase (PP-1) activities are reduced and fail to show the peak insulin stimulation observed for insulin-sensitive subjects at 10-20 minutes. All insulin resistant subjects tested showed Mn activation of trypsin treated PP-1 in the absence of azide. 6 of 10 insulin sensitive subjects, however, required azide in order to see Mn activation of PP-1. The azide appears to reverse the effects of an inhibitor of Mn activation which as been localized in the glycogen-microsomal (GM) subcellular fraction of Mn- resistant (insulin sensitive) subjects. These results suggest that an azide sensitive structure in the GM fraction of muscle is responsible for the abnormal PP-1 activity in insulin resistant subjects. Phosphorylation of the G-subunit bound to the PP-1 catalytic subunit in the GM fraction regulates PP-1 activity. Western blots of muscle extracts indicate that the G-subunit has increased immunoreactivity in insulin resistant subjects. A human homolog of rat liver PP-1 gamma cDNA was isolated from human skeletal muscle. This observation indicates that at least three PP-1 isoforms may be involved in regulation of glycogen synthase. Insulin resistant subjects have increased binding of their PP- 1 gamma isoform to the GM fraction. Although PP-1 alpha and beta also bind to the GM fraction, no differences were observed between insulin sensitive and resistant subjects. Taken together these results suggest that abnormal PP-1 activity in insulin resistant subjects may directly involve interaction between the G-subunit and PP-1 gamma. Studies of inbred mouse strains demonstrate that a diabetes susceptibility locus close to the glycogen synthase gene on mouse chromosome 7 is present in a strain of mice which have abnormal glycogen synthase activity. These results support the hypothesis that a defect in the glycogen synthase gene can contribute to the development of diabetes.